Despite the growing number of available agents designed to retard cancer growth, alkylating agents remain an important mainstay of cancer therapeutics. However, effective modulation of existing agents is necessary both to improve efficacy and to attenuate toxicity. Of the clinically useful alkylating agents, cyclophosphamide and ifosfamide are widely used for a variety of solid and hematologic malignancies and are thus important targets for manipulation. Major clinical toxicities include urotoxicity, nephrotoxicity, neurotoxicity, and, to a lesser extent, a risk of secondary leukemias. An important issue that has been raised, but not addressed directly, is the possibility that the DNA repair protein, O6-alkylguanine-DNA alkyltransferase (MGMT), protects against toxicities associated with ifosfamide and cyclophosphamide. Specific aims 1 and 2 will establish if MGMT plays a role in protecting against secondary leukemia by evaluating the repair of DNA adducts introduced by cyclophosphamide and ifosfamide by MGMT. Initially, we will determine if acrolein creates toxic/mutagenic lesions on DNA that are repaired by MGMT. These studies will be extended to the Nfl+/- mouse model of cyclophosphamide-induced leukemia. Shannon and colleagues have shown that mice heterozygous for the Nf1 gene, which encodes for a GTPase activating protein of Ras, have a higher incidence of leukemia at 15 months of age after treatment with cyclophosphamide than wild type mice. The NF1+/- mouse model of alkylating agent-induced leukemia will be crossed with MGMT-/- mice to determine if the absence of MGMT (Nfl+/-, MGMT-/-) results in an increase in the incidence of leukemia after treatment with cyclophosphamide as compared to littermates wild type for MGMT (Nf1+/-, MGMT+/+). Specific aim 3 will focus on modulating the antitumor effect of cyclophosphamide and ifosfamide without increasing toxicities associated with acrolein or chloracetaldehyde (CAA). We have identified substituted guanine analogs that increase the sensitivity of human cancer cells to phosphoramide mustard (PM), isophosphoramide mustard (IPM) but not CAA or acrolein. We will determine if the mechanism of enhancement is due to inhibition of repair of crosslinks. In this renewal, we will continue to work towards our long-term goal of developing means to protect against the toxic and mutagenic effects of alkylating agents.